Characterization of bacteria expectorated during forced salivation of the Phlebotomus papatasi: A neglected component of sand fly infectious inoculums

The infectious inoculum of a sand fly, apart from its metacyclic promastigotes, is composed of factors derived from both the parasite and the vector. Vector-derived factors, including salivary proteins and the gut microbiota, are essential for the establishment and enhancement of infection. However, the type and the number of bacteria egested during salivation is unclear. In the present study, sand flies of Phlebotomus papatasi were gathered from three locations in hyperendemic focus of zoonotic cutaneous leishmaniasis (ZCL) in Isfahan Province, Iran. By using the forced salivation assay and targeting the 16S rRNA barcode gene, egested bacteria were characterized in 99 (44%) out of 224 sand flies. Culture-dependent and culture-independent methods identified the members of Enterobacter cloacae and Spiroplasma species as dominant taxa, respectively. Ten top genera of Spiroplasma, Ralstonia, Acinetobacter, Reyranella, Undibacterium, Bryobacter, Corynebacterium, Cutibacterium, Psychrobacter, and Wolbachia constituted >80% of the saliva microbiome. Phylogenetic analysis displayed the presence of only one bacterial species for the Spiroplasma, Ralstonia, Reyranella, Bryobacter and Wolbachia, two distinct species for Cutibacterium, three for Undibacterium and Psychrobacter, 16 for Acinetobacter, and 27 for Corynebacterium, in the saliva. The abundance of microbes in P. papatasi saliva was determined by incorporating the data on the read counts and the copy number of 16S rRNA gene, about 9,000 bacterial cells, per sand fly. Both microbiological and metagenomic data indicate that bacteria are constant companions of Leishmania, from the intestine of the vector to the vertebrate host. This is the first forced salivation experiment in a sand fly, addressing key questions on infectious bite and competent vectors.


Introduction
Leishmaniases, vector-borne and poverty-related infections with a wide clinical spectrum, are regarded a major public health concern in half of the tropical/subtropical countries of the world.[1,2].By 2019, it was estimated that 0.5-0.9 million new cases of leishmaniasis occurred annually, resulting in 18,700 deaths and 1.6 million disability-adjusted life years (DALYs) lost [3].Cutaneous and mucocutaneous forms of leishmaniases (CL and MCL) had the greatest DALY increase in the last 20 years [4], and the most CL cases have been detected in eco-epidemiological "hotspot" areas within the Eastern Mediterranean region [5].In many of these areas, the outbreaks appear in 10-year intervals [6], but this trend is 1.5 times faster in some countries such as Iran [7].Zoonotic cutaneous leishmaniasis (ZCL), the most predominant and widespread form of leishmaniasis in the country, is caused by Leishmania major and chiefly vectored by the sand fly Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae) from gerbils to humans [8,9].
The epidemiology of leishmaniases is complicated and mainlly depends on the features of the parasite and sand fly species, the local ecological characteristics of the transmission areas, past and present exposure of human population to the parasite, and human behavior patterns [5,[10][11][12][13].Each leishmania species causes distinct clinical symptoms with different degrees of severity in the hosts, ranging from localized skin lesions to the reticuloendothelial system involvement.Sometimes, a parasite manifests a wide range of symptoms [12,14], but the outcome of the disease is ultimately determined by the interactions of the characteristics of the parasite, the biology of the vector, and the immune responses of the host [15,16].
Apart from the metacyclic promastigote of a sand fly, its infectious inoculum is comprised of many relevant factors derived from the parasite and vector [17].The Leishmania-derived factors mainly include proteophosphoglycans and exosomes, which respectively contribute to disease exacerbation by modulating early innate pathways involved in wound response, as well as to the alteration of cell recruitment/behavior patterns.As for vector-derived infection enhancers, both sand fly saliva and gut microbiota play a role.Sand fly salivary proteins are beneficial for the establishment and promotion of infection.It has been shown that bacteria in the intestinal tract of a sand fly are contributing factors to the development of the Leishmania parasite inside the midgut and to the enhancement of parasite-derived infection during coegestion to the vertebrate host [18][19][20][21].
Female vector sand flies feed on vertebrate blood and natural sugars such as sap, nectar, fruits, or secretions Homoptera [22].They salivate during both types of feeding.In the case of sugar meals, saliva is secreted to break down oligosaccharides with α-glucosidase and starch with amylase, as well as to dilute highly concentrated sugar solutions to facilitate ingestion [23].In contrast, during blood feeding, the sand fly uses the pharmacologically active components of saliva, with anti-hemostatic, anti-inflammatory and immunomodulatory properties that facilitate the blood-meal intake with the least host resistance [24].Phlebotomines are basically pool feeders or "telmophages", and they suck blood and lymph from small wounds they make on the bite site [25].During salivation, the vector insects may release microorganisms associated with the salivary glands and digestive tract, i.e. viruses, bacteria, and other pathogens, into the feeding substrates [17,18,26,27].
Studies have recently shown that bacteria in the sand fly's intestine can affect the establishment and pathogenesis of the Leishmania parasite in the vertebrate host by modulating the immune system [18,20].This behavior can be deduced simply by comparing the needle infection model with the transmission of pathogens through bites [19].Now, the following questions are raised: what kind of and how many bacteria are loaded during a P. papatasi sand fly salivation?The current research was designed and conducted to answer these basic questions.

Ethics statement
The ethical considerations of this study were approved by the institutional animal and human ethical committee under national and international standards with the ethical code: IR.PII.REC.1399.027by Pasteur Institute of Iran.

Sand fly collection
Live adult sand flies were captured using a car trap from different areas of Isfahan Province during June and July 2020.A car trap is a newly parked light-colored passenger vehicle used to attract sand flies in the vicinity of rodent burrows at sunset.Following landing on the car, the sand flies were collected using a mouth aspirator and flashlight.The gathered flies were transferred to the Entomology Laboratory of the Department of Parasitology at the Pasteur Institute of Iran and kept in metal cages and inside a net, while maintaining humidity and providing with 10% sucrose.After the flies were acclimated to the laboratory conditions (24-27˚C, 80% RH, and 14:10 [L:D]), and the transportation stress was gone, they were starved of sugar for 24 hours, prior to the forced salivation experiment.

Forced salivation experiment
This assay is actually the modified method of mosquito vector competence for arboviruses [28].In brief, the sand flies were taken out from the cage with an aspirator and anesthetized by being placed at -20˚C for 2 minutes.To keep anesthesia during the experiment, the flies were transferred to a Petri dish on an ice tray and then examined individually on a clean slide under a stereo microscope.Their wings and legs were cut using tweezers and fine needles, to prevent any movement.As depicted in the Fig 1, the proboscis of each insect was placed in the entrance of a yellow pipette tip with the help of the same tools.Insects were offered sugar meals (10 μl of 10% sucrose solution supplemented with 2% red food colorant) through the yellow pipette tips.The tips, from their wide and narrow sides, were fixed on a glass plate (40 cm × 30 cm) with playdough and a double-sided stick, respectively.After inserting all the proboscises into the tips, the glass plate containing the test specimens was left intact for about one hour in laboratory conditions and away from any air flow, to allow sand flies feeding.Sand flies with red solution in their bodies (complete or partial) were considered as insects with forced salivation (Fig 1A -1E).

Culture-dependent identification of bacteria egested during forced salivation
Sand flies whose bodies turned red, i.e. those who had salivated and fed, were transferred to a drop of sterile PBS on a glass slide and micro-dissected under a stereo microscope.The dissected guts and the rest of the sugar solution in the yellow pipette tips were separately transferred to falcons containing 5 ml of brain-heart infusion (BHI) broth.The head and the ends of the abdominal segments were mounted on a slide for morphological identification.Each culture medium was named based on the specific code of the corresponding glass slides incubated at 100 rpm at 37˚C for 24-48 hours.The opaque media were considered positive specimens and subcultured at above conditions in a BHI agar medium overnight.After obtaining phenotypically unique colonies, pure isolates were photographed.Cultures of pure single colonies were stored in a glycerol containing BHI broth at -80˚C, until molecular surveys.In each test period, possible contaminant bacteria of tips, needles, and sand fly cuticles and also sugar meal were cultured separately as negative controls of the experiment.
For molecular identification of bacteria, the genomic DNA of pure isolates was prepared using phenol:chloroform:isoamyl alcohol (24:25:1 v/v) method, in line with a previously described protocol [29].Five hypervariable (V1-V5) regions of the 16S rRNA gene of bacteria were targeted to amplify ~800 bp of the gene.The gene amplification and sequencing were carried out based on methods described in the literatures [30,31].Bacterial identities were determined with focusing on the results of a BLASTn search of 16S rRNA sequences against nucleotide sequences obtained from the NCBI [32] and leBIBI [33] databases.Sequences obtained through Sanger sequencing were submitted into the GenBank database.
To determine the phylogenetic position of bacteria found in the sand fly saliva, we compared 404-431 bp of 16S rRNA fragments of bacteria with the representatives of environmental and pathogenic counterparts validated in List of Prokaryotic names with Standing in Nomenclature (LPSN) [44], using maximum likelihood tree construction method.

Quantification of bacteria load during sand fly salivation
Four pipette tips, containing sand flies' saliva, were used to compute the number of bacteria transmitted during fly bites through the smear preparation (n = 2) or optical density (OD) measurement (n = 2).The number of 16S rRNA gene copies for bacteria with more than 25 reads was also used as a basis for determining the number of bacteria egested into each pipette tip.In this regard, 16S rRNA gene copy counts for each bacterium identified in this study were first retrieved from the website of the University of Michigan Center for Microbial Systems [45].Then the number of reads calculated for each bacterium was divided by the average number of 16S rRNA gene copies determined for that taxon in the above website.Bacteria with unknown identity, specified as "uncultured", were not included in the calculations.

Forced salivation experiment results
A total of 1891 P. papatasi were caught from Habib-Abad (n = 1003) and Matin-Abad (n = 888) Counties in the hyperendemic focus of ZCL in Isfahan Province, the centr of Iran.A number of 224 female sand flies were examined for forced salivation, which the experiment was successful in 99 (44.20%)sand flies with a range of 21.67%-55.00%(Table 1; Fig 1).

Culture-dependent identification of bacteria
Seventy-seven samples from saliva-gut mutual set were used for bacterial culture, single colony preparation, DNA extraction, and PCR-sequencing.About 800 bp of the 16S rRNA gene of bacteria was successfully sequenced, and the consensus data were deposited in the GenBank under the accession numbers ON314273-ON314424.The sequence analysis determined the presence of 8 families, 8 genera, and 14 species of bacteria in the saliva-gut communities of sand flies.Enterobacter hormaechei, a component of the E. cloacae complex, was detected as the most abundant bacterium in the gut (n = 60) and saliva (n = 61) of sand flies.Two bacteria of Enterobacter hormaechei and Priestia flexa were found in the cuticle and needle controls, respectively (Fig 2 , Table 2).

Culture-independent identification of bacteria
Three individual saliva specimens failed during the quality control (QC) measurement for next-generation sequencing (NGS) analysis.Also, among pooled pairs, only saliva-gut cross pairs were worth analyzing; therefore, our study focused on one of these pairs with successful QC.Illumina HiSeq sequencing platform yielded a total of 122,730 (58154 for gut/64576 for saliva) bacterial 16S rRNA gene sequences from reciprocal saliva-gut assemblages after trimming, measuring QC, and removing plastid/mitochondrial sequences.The identified operational taxonomic units (OTUs) were divided into 13   more than 80% of the saliva microbiome.Interestingly, 16S rRNA gene sequences of some bacteria were found to be more in saliva than intestine (Table 3; Fig 3).The high-throughput sequences of 16S rRNA gene fragments were deposited in the NCBI database under Sequence Read Archive (SRA) with Bioproject identification number PRJNA861709.

Quantification of bacteria load during sand fly salivation
The model used for bacteria quantification displayed the minimum number of bacteria in the body of sand flies and those that are egested during feeding as 41,205 (� x = 8,241 per sand fly) and 45,763 (� x = 9,152 per sand fly) cells, respectively.The number of bacteria egested was 1.1 times the number of bacteria in the guts of the sand flies.Neither smear preparation nor OD measurement were successful in quantifying the bacterial load.

Discussion
In this research, the quantity and quality of bacteria were studied in the saliva of the sand fly P. papatasi, the main vector of ZCL.Culture-dependent and culture-independent methods identified E. cloacae complex members and Spiroplasma as dominant taxa, respectively (Tables 2  and 3).The model used to quantify bacteria revealed that each sand fly left at least 9,152 different bacterial cells in the feeding substrate.As emphasized in recent investigations, the abovementioned bacteria not only can be potential pathogens in the vertebrate host but also may cause more serious consequences when accompanied by the L. major parasite [18,20].
It is thought that all aspects of leishmaniasis, as one of the most important tropical/subtropical diseases of the world, have been thoroughly investigated.However, besides the main components of the disease (vector, pathogen, reservoir, and host) and their interaction, other factors are involved in the outcome of leishmaniasis, many of which are unknown or little known [20].In this regard, the role of bacteria-especially those transmitted during vector bites-in the pathogenesis of vector-borne pathogens has largely been overlooked.In line with the studies by Dey et al. and Amni et al., the results of our study identified bacteria as an important part of the life cycle of leishmaniasis (Tables 2 and 3).Bacteria found in an infectious bite can be effective in the wound formation and worsening of lesions [18], as well as may role play in the continuation of the infection and even recovery of the disease [20].Owing to the fact that the microbiota of sand fly vectors are affected by intrinsic and extrinsic factors, including the microclimate of the location [46,47], wounds that do not heal [48] or in which the symptoms progress or lesions spread [1,49,50] can be attributed to the variation in microbiota of the sand flies.Forced saliva assay is, by definition, forcing the insect to secrete saliva with the purpose of basic and practical-field experiments (Fig 1).The method are typically used to measure the mosquitoes competence in arboviral diseases [28,[51][52][53][54], to assess viral load in vector saliva [54] or to characterize the immunomodulatory properties of salivary proteins from different mosquito species [27].In case of malaria, the forced salivation method is used to estimate extrinsic incubation period in individual mosquitoes and study Plasmodium-Anopheles interactions [55], It is also used in monitoring and control programs as a rapid detection method for the presence of infectious mosquitoes capable of transmitting malaria [26].In more limited cases, this technique can be used to understand the interactions between pathogens in coinfections, as reported in previous studies [56][57][58].To the best of our knowledge, this is the first study conducted using forced salivation assay in sand flies to investigate bacteria released during salivation, which can help discriminate proven vectors of diseases by identifying Leishmania metacyclic parasites and also viruses in their infectious saliva.
In this study, the average success rate of forced salivation was found to be about 44% (Table 1), which seems to be a low success rate.The duration of starvation before testing the sand flies had a direct effect on their salivation success rate.In the current study, we used fieldcaught specimens, which presumably be more successful with insectary populations.However, the results of the present study are very valuable from the point of view that the sand flies used were from the ZCL hyperendemic focus of Isfahan Province, where L. major is being transmitted.
The most common method of measuring the abundance of microbes of different niches is to gather DNA samples and sequence a specific gene such as the 16S rRNA as a "barcode gene" from those samples [59].Considering several assumptions, it was possible to estimate the frequency of microbes in P. papatasi saliva based on 16S Illumina sequencing and 16S genomic copy number (Table 3).The applied method has been shown to be valid in estimating microbial communities in numerous habitats [60][61][62][63][64].The copy number of 16S rRNA gene can vary from 1 to more than 15 [65], and large copy number variation can cause bias in the estimation of relative abundance of cells and incorrect qualitative interpretations [59].Therefore, in this study, the average number of copies for each bacterial genus was considered to minimize this error (Table 3).Also, in this study, the V1-V5 regions of the 16S rRNA gene, showing the least intragenomic heterogeneity in bacteria [66], were targeted, which is another strength of the study.
Sanger sequencing and NGS methods respectively identified E. cloacae and Spiroplasma as the predominant microbes in P. papatsi saliva (Tables 2 and 3).Enterobacter cloacae is a commensal bacterium in the digestive tract of humans and animals as well as insects of medical importance [67], which its natural circulation has been proven among the ZCL partners in the hyperendemic focus of Isfahan Province [30], and after successful genetic manipulation [68] and evaluation of its stability in sugar bait [69], has passed the preliminary experiments to reduce pathogen transmission in the platform of a paratransgenesis approach.Considering the widespread of bacterium in nature and acting as an opportunistic microbe [67], future studies should also pay attention to the possibility of causing infection by E. cloacae in the vertebrate host during the bite of the vectors.
Spiroplasmas are of special interest due to their unique morphology, motility, and lifestyle, as well as their economic and medical significance as pathogens of plants, insects, and vertebrates [70,71].The well-known species are S. poulsonii, reproductive manipulator of flies and also S. citri and S. kunkelii, the plant pathogens [72,73], which their sequences clustered within the Spiroplasma clade found in this study.Spiroplasma has been detected in several fly, mosquito, and sand fly species [74][75][76][77].A study on sand flies revealed the presence of this bacterium in a quarter of females but not in males, which probably specifies the occurrence of a male-killing phenomenon [77].Although the possibility of infection of humans and other vertebrates with Spiroplasma species in natural conditions is largely unknown, the possibility of transmission of these bacteria by blood-sucking insects is not out of mind.Sand flies secrete saliva while feeding on both sugar [23] and blood [24] meals.The initial strategy of the current study was to identify saliva bacteria during blood feeding of sand flies; however, due to the refusal of field-caught flies to feed on blood, the study was shifted to a sugar meal.It is thought that the process of saliva secretion during feeding from two types of food sources is different, which its investigation requires a special methodology.
While culture-independent Illumina HiSeq sequencing, compared to Sanger method, significantly detected more bacterial taxa (Tables 2 and 3), the dominant taxa from the Illumina data were not identified by traditional culture-dependent methods.All the bacteria observed in the sand fly saliva were also found in the gut (Table 3; Fig 3); however, the culture-independent method determined the number of the egested bacteria more than in the intestine.This observation, in fact, highlights the importance of released bacteria in modulating the vertebrate host's immune system, pathogen establishment, and pathogenesis.
In previous studies, the average infectious dose of Leishmania parasite transmitted by the vector to the vertebrate host was determined to be 1,000 parasites for each sand fly [78,79].However, the co-occurrence of the parasites with intestinal bacteria as sand fly infectious inoculum has relatively been disregarded.The model studied herein not only verified the transmission of bacteria during the bite but also presented their number to be nine times that of infectious parasites in the vector.The numbers obtained here may not be definitive and may change according to the study method and nutritional physiology of the vector.It is also possible that the forced feeding process egestes more bacteria than normal which should wait for more details in future studies.Moreover, studies on multiple Leishmania-sand fly-host combinations have uncovered that pre-exposure of hosts to sand fly bites confers significant protection against Leishmania infection [80].In the distant years, this pre-immunity was attributed to at least 20 diverse salivary proteins with anti-hemostatic and anti-inflammatory properties [24], but studies in recent years have emphasized the role of bacteria transmitted along with saliva [18,20].
Modulation of the host's immune system may be true for any bacteria identified in the current study.However, it is likely to be more significant for Acinetobacter and Corynebacterium, which were found with high phylogenetic diversity in saliva (S3 and S7 Figs), and whose immunomodulatory abilities have been previously reported [81,82].

Conclusion
The absence of a vaccine and treatment failure in leishmaniasis provides many motivations to better understand the factors cause the inflammatory response.The vector gut microbiota, as a major player, can act an important role in modulating the host immune responses for the establishment and spread of a pathogen and even recovery from the disease.The present study determined the type and number of bacteria left in the feeding substrate during the forced salivation of P. papatasi sand flies caught from the hyperendemic focus of ZCL in Isfahan Province.With the quantification of metagenomic traits, the possibility of exploiting bacteria in the explanation of the infectious inoculum of the sand fly vector became possible.Collectively, the findings of this study can improve our insight into measuring the effect of vector-derived bacteria on the improvement or deterioration of leishmaniasis.